A liver tumor derived from a B6C3Fl mouse was shown to contain a dominant acting transforming gene as determined by transfection assay. As a novel means of isolating this transforming gene, a cDNA library was constructed and inserted into a cDNA expression vector engineered in this laboratory. This cDNA expression library was transfected into NIH/3T3 cells. Transforming foci were isolated and cDNA containing plasmids were rescued from the cells by a process of specific restriction enzyme cleavage, ligation, and transformation into E. coli cells. The various plasmids isolated were transfected into NIH/3T3 cells to determine which isolated cDNA had transforming activity. The transforming activity was associated with a plasmid containing a 4.0-kb cDNA insert. Subsequent gene sequence analysis demonstrated that this transforming gene was a truncated B-raf gene. This truncated B-raf had a constitutively activated kinase activity, similar to a truncation previously identified in a c-raf oncogene. Hybridization studies of this isolated B-raf truncated gene to the DNA from the original mouse liver tumor verified that this specific B-raf truncation was present in the original tumor.